The delivery of small molecules, oligonucleotides, and proteins through biological membranes is a major challenge facing therapy and validation paradigms. It has recently been established that transducing peptides derived from Antennapedia, TAT-HIV, and VP22 can penetrate biological membranes, act as cargo vehicles, and target to specific subcellular compartments. Here we show the identification of a nuclear localization sequence (NLS) within human Period 1 (hPER1) circadian protein that functions as a transducing peptide. More importantly, using database mining, we have uncovered additional transducing peptides embedded within the NLS's of other proteins and extend the number of gene-encoded transducing peptides from 3 to 14. Our data suggest that transducing peptides are found within NLS's and are prevalent, diverse, and distributed widely throughout the genome. It is well established that certain extracellular and intracellular proteins are targeted to specific organelles within a cell, transmembrane or secreted from the cell. The biological mechanisms by which intracellular protein targeting occurs continues to be characterized, but is well recognized that one mechanism for localization occurs by virtue of specific leader sequence contained within the protein of interest, or intraprotein sequence. Localization of proteins within selected cellular organelles is aided by specific targeting sequences. A number of nuclear localization sequences (NLSS) have been identified in proteins that permit the protein to be transported or otherwise pass from the cytoplasm into the nuclear membrane.
Fusion proteins containing the targeting sequence and another, otherwise non-targeted protein, are localized in the selected cellular organelle depending on the targeting sequence selected. For example, Ferullo, J. M. and Paget, E. FR 279695, disclose selective compartmentalization of an hydroxyphenylpyruvate dioxygenase (HPPD) fused to a signal sequence directing the enzyme to a cellular compartment other than the cytosol, e.g., a vacuole. Similarly, WO 0147950 (Wehrle-Haller, Bernhard M.; Imhof, Beat A) identify a new determinant responsible for basolateral targeting and prolonged exposure of cell-surface-anchored growth factors at cell surfaces. The signal is a mono-leucine dependent basolateral sorting signal consisting of the amino acid sequence X1h2X3h4Lp5p6, wherein: X1 represents a polar amino acid residue or alanine, h2 represents any hydrophobic amino acid residue, X3 represents any amino acid residue, h4 represents any hydrophobic amino acid residue, except leucine and isoleucine, L represents a leucine residue, p5 represents any polar amino acid residue, and p6 represents any polar amino acid. Richardson, A. E., et al., Plant J. (2001), 25(6), 641-649 describe manipulation of the enzyme aspergillus phytase to include the signal peptide sequence from the carrot extensin gene. The resulting fusion protein was only effective when secreted as an extracellular enzyme into the adjacent soil, and resulted in a 20-fold increase in total root phytase activity in transgenic lines and subsequent improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. WO 0132894 (Lok, S.) disclose use of the signal anchor domain sequences of type II cell surface proteins to anchor recombinant proteins into surface of transfected cells. A characteristic feature of type II cell surface proteins is that they are held within the cellular membrane by a single hydrophobic transmembrane domain and are oriented with their C-terminus outside the cell.
More recently, a few proteins have been identified which are capable of passing through the cellular membrane without requiring active transport mechanisms or ‘pores’. It is recently established that membrane penetrating peptides (MPPs, also known as protein transduction domain, “PTD”) derived from Antennapedia, TAT, and VP22 can penetrate biological membranes and target to specific subcellular compartments. None of these previously disclosed proteins are derived from mammalian proteins. The present invention is directed to the discovery that polypeptides derived from mammalian or yeast proteins nuclear localization sequences (NLSs) or overlapping with NLS's are capable of acting as MPPs, and identification of a specific polypeptide sequences capable of penetrating cellular membranes, even when conjugated to large proteins, such as biologically active proteins, or other organic compounds.
Nuclear transport is essential to a number of biological processes including gene expression and cell division, as well as to viral replication, tumorigenesis and tumor cell proliferation. The mechanism of nuclear transport has only recently been characterized in detail and has been shown to involve a number of discrete steps. Proteins that are destined to be transported into the nucleus contain within their amino acid sequence a short stretch of amino acids termed a nuclear localization sequence (“NLS”). These sequences may occur anywhere within the amino acid sequence and are typically four to about eight amino acids. These sequences are generally basic (i.e., positively charged) in nature, however, there has been no consensus sequence identified. Thus, there is a wide variety of these sequences that appear to be specific for particular proteins.
Within the cell, these NLSs may be either masked or unmasked by accessory proteins or by conformational changes within the NLS-containing protein. An NLS may be masked because it is buried in the core of the protein and not exposed on the surface of the protein. Unmasking of NLSs, and nuclear translocation of cytoplasmic proteins may be triggered by phosphorylation, dephosphorylation, proteolytic digestion, subunit association or dissociation of an inhibitory subunit, or the like. Accordingly, the masking and unmasking of NLSs provides a mechanism by which the transport of these cytoplasmic proteins into the nucleus may be regulated. For example, the transcription factor NF-AT contains nuclear localization sequences which allow NF-AT to translocate to the nucleus in the presence of intracellular calcium, but which are shielded by forming intramolecular associations with other domains in the NF-AT polypeptide in the absence of calcium.
Lee, H. C. and Bernstein, H. D. Proc. Natl. Acad. Sci. U.S.A. (2001), 98(6), 3471-3476 studied the mechanism involved for presecretory proteins such as maltose binding protein (MBP) and outer membrane protein A (OmpA) that are targeted to the E. coli inner membrane by the molecular chaperone SecB, in contrast to the targeting of integral membrane proteins by the signal recognition particle (SRP). The authors found that replacement of the MBP or OmpA signal peptide with the first transmembrane segment of AcrB abolished the dependence on SecB for transport and rerouted both proteins into the SRP targeting pathway.
Some proteins contain cytoplasmic localization sequences (CLS), or nuclear export sequences, which ensure the protein remains predominantly in the cytoplasm. For example, Hamilton, M. H. et al., J. Biol. Chem. (2001), 276(28), 26324-26331 demonstrate that the ubiquitin-protein ligase (E3), hRPF1/Nedd4, a component of the ubiquitin-proteasome pathway responsible for substrate recognition and specificity, is capable of entering the nucleus, but the presence of a functional Rev-like nuclear export sequence in hRPF1/Nedd4 ensures a predominant cytoplasmic localization. The cytoplasmic domains of many membrane proteins contain sorting signals that mediate their endocytosis from the plasma membrane.
Heineman, T. C. and Hall, S. L. Virology (2001), 285(1), 42-49 studied three consensus internalization motifs within the cytoplasmic domain of VZV gB and determined that internalization of VZV gB, and its subsequent localization to the Golgi, is mediated by two tyrosine-based sequence motifs in its cytoplasmic domain. In mammalian cells and yeasts, amino acid motifs in the cytoplasmic tails of transmembrane proteins play a prominent role in protein targeting in the early secretory pathway by mediating localization to or rapid export from the endoplasmic reticulum (ER). Hoppe, H. C. and Joiner, K. A. Cell. Microbiol. (2000), 2(6), 569-578.
The mammalian endopeptidase, furin, is predominantly localized to the trans-Golgi network (TGN) at steady state. The localization of furin to this compartment seems to be the result of a dynamic process in which the protein undergoes cycling between the TGN and the plasma membrane. Both TGN localization and internalization from the plasma membrane are mediated by targeting information contained within the cytoplasmic domain of furin. Voorhees, P., et al., EMBO J. (1995), 14(20), 4961-75 report that there are at least two cytoplasmic determinants that contribute to the steady-state localization and trafficking of furin. The first determinant corresponds to a canonical tyrosine-based motif, YKGL (residues 758-761), that functions mainly as an internalization signal. The second determinant consists of a strongly hydrophilic sequence (residues 766-783) that contains a large cluster of acidic residues (E and D) and is devoid of any tyrosine-based or di-leucine-based motifs. This second determinant is capable of conferring localization to the TGN as well as mediating internalization from the plasma membrane.
The trans-Golgi network (TGN) plays a central role in protein sorting/targeting and the sequence SXYQRL can by itself confer significant TGN localization. Wong, S. H., and Hong, W. J. Biol. Chem. (1993), 268(30), 22853-62 report detailed mutagenesis of the 32-residue sequence of TGN38, an integral membrane protein confined mainly to the TGN, and determined that the Ser, Tyr, and Leu residues at positions 23, 25, and 28, respectively, are essential for TGN localization. When the cytoplasmic 32-residue sequence of TGN38 was fused to the ecto- and transmembrane domains of glycophorin A (a surface protein), the resulting chimeric protein was localized to the TGN.
It is well recognized that certain proteins are either only active in a specific organelle, or are capable of different functions depending on their localization. For example, appropriate subcellular localization is crucial for regulation of NF-κB function. Huang, T. T., et al., Proc. Natl. Acad. Sci. U.S.A. (2000), 97(3), 1014-1019, show that latent NF-κB complexes can enter and exit the nucleus in preinduction states and identified a previously uncharacterized nuclear export sequence in residues 45-54 of IκBα that was required for cytoplasmic localization of inactive complexes. It appears that NF-κB/IκBα complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export and that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-κB activation by extracellular signals.
Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. In combination with Ran, two other soluble factors are thought to be absolutely required to mediate the nuclear import of a protein containing a classical or basic NLS into the nucleus. The first is karyopherin/importin α (Kap α), which binds a classical NLS and then forms a complex with karyopherin/importin β1 (Kappβ1). Adam, S. A., and Gerace, L. (1991) Cell 66, 837-847; Görlich, D., et al. (1994) Cell 79, 767-778; Moroianu, J., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2008-2011; Radu, A., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 1769-1773; Görlich, D., et al. (1995) Curr. Biol. 5, 383-392; Chi, N. C., et al. (1995) J. Cell Biol. 130, 265-274. Kap β1 interacts with nuclear pore complex (NPC) proteins and appears to mediate movement of the import complex through the NPC via these interactions. Rexach, M., and Blobel, G. (1995) Cell 83, 683-692; Radu, A., Blobel, G., and Moore, M. S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 1769-1773; Iovine, M. K., Watkins, J. L., and Wente, S. R. (1995) J. Cell Biol. 131, 1699-1713; Radu, A., Moore, M. S., and Blobel, G. (1995) Cell 81, 215-222. Another protein, p10/NTF2, has also been implicated in nuclear import, but its function may only be to take Ran into the nucleus, where it is subsequently needed to disassemble an incoming import complex. Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10212-10216; Paschal, B. M., and Gerace, L. (1995) J. Cell Biol. 129, 925-937; Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M., and Görlich, D. (1998) EMBO J. 17, 6587-6598; Smith, A., Brownawell, A., and Macara, I. G. (1998) Curr. Biol. 8, 1403-1406.
Although there is only one Kap a homologue in yeast (SRP1 or Kap60), vertebrate cells contain a number of proteins that can bind a classical NLS and share sequence homology (see Ref. Nachury, M. V., Ryder, U. W., Lamond, A. I., and Weis, K. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 582-587, and references therein). These proteins have been given a variety of names but can be grouped into three major families. The Kap α1 family contains the human protein NPI-1/importin α1/karyopherin α1/Rch2/hSRP1 and a second related protein importin α6, in addition to the mouse S2 protein. Moroianu, J., et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2008-2011; Cortes, P., et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7633-7637; O'Neill, R. E., et al., (1995) J. Biol. Chem. 270, 22701-22704; Kohler, M., et al., (1997) FEBS Lett. 417, 104-108; Tsuji, L., et al., (1997) FEBS Lett. 416, 30-34. The second family, Kapα2, contains human Rch1/hSRP1/importin α2/karyopherin α2 and the mouse protein pendulin/PTAC 58. Görlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Cell 79, 767-778; Cuomo, C. A., Kirch, S. A., Gyuris, J., Brent, R., and Oettinger, M. A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6156-6160; Kussel, P., and Frasch, M. (1995) Mol. Gen. Genet. 248, 351-363; Imamoto, N., Shimamoto, T., Takao, T., Tachibana, T., Kose, S., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. (1995) EMBO J. 14, 3617-3626; K., Mattaj, I. W., and Lamond, A. I. (1995) Science 268, 1049-53. The third family, Kapα3, consists of the two human proteins, QIP-1/importin α3 and KPNA3/hSPR1 γ/hSRP4, and the mouse proteins Q1 and Q2. Nachury, M. V., et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 582-587; Kohler, M., et al., (1997) FEBS Lett. 417, 104-108; Tsuji, L., et al., (1997) FEBS Lett. 416, 30-34; Takeda, S., et al., (1997) Cytogenet. Cell Genet. 76, 87-93; Seki, T., et al., (1997) Biochem. Biophys. Res. Commun. 234, 48-53; Miyarnoto, Y., et al., (1997) J. Biol. Chem. 272, 26375-26381. Each of these classes share about 50% homology with each other and to the yeast SRP1, and each of these mammalian proteins has been shown to be capable of mediating the import of one or more classical NLS-containing proteins. Nachury, M. V., et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 582-587; Sekimoto, T., et al., (1997) EMBO J. 16, 7067-7077; Nadler, S. G., et al., (1997) J. Biol. Chem. 272, 4310-4315; Prieve, M. G., et al., (1998) Mol. Cell. Biol. 18, 4819-4832.
Stat-1 import is mediated by Kapα1/NPI-1 but not Kapα2/Rch1, but activated Stat-1 appears to bind to a COOH-terminal region of Kapα1 distinct from the NLS binding Armadillo repeats. The binding differences of the different Kapαs to RCC1 observed appear to be due solely to the NLS on RCC1 and therefore probably due to the NLS binding region of Kapα3. Sekimoto, T., et al., (1997) EMBO J. 16, 7067-7077. Kamei, Y., et al., (1999) J. Histochem. Cytochem. 47, 363-372 showed that, in mice, the Kapα3 homologue is expressed in many tissues and theorized that Kapα3 may play a role in importing “a limited number of unique karyophilic proteins, such as helicase Q1.” The results provided by Talcott, B. and Moore, M. S., 2000 J Biol Chem, 275(14) 10099-10104 suggest that RCC1 should be included in the group of proteins that use Kapα3 to mediate their nuclear import.
U.S. Pat. No. 6,191,269 teaches the existence of a nuclear localization sequence contained within the cDNA sequence of the N-terminal IL-1 alpha propiece, T76-NGKVLKKRRL (SEQ ID NO:1), which had characteristics of a nuclear localization sequence (NLS) and could mediate nuclear localization of the propiece (Stevenson et al. (1997) Proc. Natl. Acad. Sri. USA 94:508-13. Introduction of the cDNA encoding the N-terminal IL-alpha propiece into cultured mesangial cells resulted in nuclear accumulation (Stevenson et al. id).
U.S. Pat. No. 5,877,282 teaches that the antennapedia homeodomain signal sequence peptide is the amino acid sequence RQIKLWFQNRRMKWKK (SEQ ID NO:2); the fibroblast growth factor signal sequence peptide is AAVALLPAVLLALLA (SEQ ID NO:3); the HIV Tat signal sequence peptide is the amino acid sequence CFITKALGISYGRKKRRQRRRPPQGSQTH (SEQ ID NO:4).
Schwartze, S. R., et al., Science 285:1569-1572 (1999) report delivery of an ip injected reporter protein, 116 kD beta-galactosidase, as a TAT fusion protein into tissues and across the blood-brain barrier. Schwartze used an 11 amino acid protein transduction domain (PTD) derived from HIV tat protein with an N-terminal fluorescein isothiocyanate (FITC)-Gly-Gly-Gly-Gly (SEQ ID NO:5) motif. The authors report that earlier attempts to transduce beta-Gal chemically cross-linked to the TAT PTD resulted in sporadic and weak beta-Gal activity in a limited number of tissues. They speculate that the improved transduction was due to the in-frame fusion and purification strategy used.
Nuclear localization of IFNγ is mediated by a polybasic NLS in its C terminus, which is required for the full expression of biological activity of IFNγ, both extracellularly and intracellularly. Subramaniam, Prem S., et al., J. Cell Sci. (2000), 113(15), 2771-2781. This NLS is thought to play an integral intracellular role in the nuclear translocation of the transcription factor STAT1α activated by IFNγ because treatment of IFNγ with antibodies to the C-terminal region (95-133) containing the NLS blocked the induction of STAT1α nuclear translocation, but these antibodies had no effect on nuclear translocation of STAT1α in IFNα treated cells. A deletion mutant of human IFNγ, IFNγ(1-123), which is devoid of the C-terminal NLS region was biologically inactive, but was still able to bind to the IFNγ receptor complex on cells with a Kd similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFNγ(1-123) to initiate the nuclear translocation of STAT1α, which is required for the biological activities of IFNγ following binding to the IFNγ receptor complex. A C-terminal peptide of murine IFNγ, IFNγ(95-133), that contains the NLS motif, induced nuclear translocation of STAT1α when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1α nuclear translocation. In cells activated with IFNγ, IFNγ was found to as part of a complex that contained STAT1α and the importin-α analog Npi-1, which mediates STAT1α nuclear import. The tyrosine phosphorylation of STAT1α, the formation of the complex IFNγ/Npi-1/STAT1α complex and the subsequent nuclear translocation of STAT1α were all dependent on the presence of the IFNγ NLS.
The peptide representing amino acids 95-132 of IFN (IFN-γ (95-132)), containing the polybasic sequence 126RKRKRSR132 (SEQ ID NO:6), was capable of specifying nuclear uptake of the autofluorescent protein, APC, in an energy-dependent fashion that required both ATP and GTP. Nuclear import was abolished when the above polybasic sequence was deleted. Subramaniam, P., et al., 1999 J Biol Chem 274(1) 403-407. A peptide containing the prototypical polybasic NLS sequence of the SV40 large T-antigen was also able to inhibit the nuclear import mediated by IFN-γ (95-132), suggesting that the NLS in IFN may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Intact IFN-γ, when coupled to APC, was a iso able to mediate its nuclear import, and this nuclear import was blocked by the peptide IFN-γ (95-132) and the SV40 T-NLS peptide, suggesting that intact IFN-γ was also transported into the nucleus through the Ran/importin pathway.
Nuclear proteins are imported into the nucleus through aqueous channels that span the nuclear envelope called nuclear pore complexes (NPCs). Although ions and molecules less than ˜20-40 Da can diffuse passively through the nuclear pore complexes, larger proteins are transported by saturable pathways that are energy- and signal-dependent. The signals that specify nuclear protein import (NLSs) are commonly short stretches of amino acids rich in basic amino acid residues, although other classes of NLSs have been described recently. The initial step in the import of proteins containing basic amino acid-type NLSs occurs in the cytosol, where the NLS-containing proteins are bound to a receptor (variously called the NLS receptor, importin α, and karyopherin (13). The substrate-receptor complex then associates with the cytoplasmic face of the nuclear pore complexes, and with the participation of other cytosolic factors, is transported through a gated channel in the nuclear pore complexes to the nuclear interior. The in vivo events of NLS-mediated nuclear import can be duplicated in an in vitro system using digitonin-permeabilized cells supplemented with cytosolic extracts and ATE (14). Transport in this in vitro assay is blocked by the same inhibitors that block in vivo import, is rapid, and is easily quantified.
The NLS the sequence NYKKPKL (SEQ ID NO:7) in the N-terminus of fibroblast growth factor (FGF)-1, the precursor for acidic FGF, has been proposed to affect the long term activities of FGF-1 through its function as a nuclear translocation signal or its role in stabilization of the structure required to sustain binding and activation of the transmembrane receptor kinase. Luo, Y., et al., J. Biol. Chem. (1996), 271(43). 26876-26883. For example, concurrent with a marked increase in dependence on exogenous heparin for optimal activity, sequential deletion of residues in the NYKKPKL (SEQ ID NO:7) sequence in FGF-1 resulted in a progressive loss of thermal stability, resistance to protease, mitogenic activity, and affinity for the transmembrane receptor. The largest change resulted from deletion of the entire sequence through the lysine-leucine residues. In the presence of sufficiently high concentrations or heparin, the deletion mutants exhibited mitogenic activity equal to wild-type FGF-1.
Although FGF-1 contains a nuclear translocation sequence (NTS), nuclear translocation requires an exogenous and not an endogenous pathway. The NTS of FGF-1 NYKKPKL (SEQ ID NO:7), is able to direct the expression of the bacterial β-galactosidase (βgal) gene to the nucleus of transfected NIH 3T3 cells, but this NTS is unable to target either FGF-1 itself of a FGF-1-βgal fusion protein into tire nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Zhan, X., et al., Biochem. Biophys. Res. Commun. (1992), 188(3), 982-91.
Interferon-γ (IFN-γ), a protein that uses the Jak-Stat pathway for signal transduction, translocates rapidly to the nucleus in cells treated extracellularly with the cytokine. An NLS has been identified and characterized in the C-terminus of human and murine IFN-γ. Larkin, J., et al., J. Interferon Cyokine Res. (2001), 21(6), 341-348 report that human IFN-γ (HuIFN-γ) contains a second NLS at an upstream site. The primary sequence, analogous with the NLS sequence identified in murine IFN-γ, representing amino acids 122-132 of HuIFN-γ was capable of mediating the nuclear import of the autofluorescent protein allophycocyanin (APC) in an energy-dependent manner. The second sequence, representing amino acids 78-92 of HuIFN-γ, was also capable of mediating the nuclear import of APC in an energy-dependent manner but to a greatly reduced extent. The nuclear import of both sequences conjugated to APC was strongly blocked by competition with unconjugated HuIFN-γ(122-132). Competition by the sequence HuIFN-γ(78-92) effectively blocked the import of APC-conjugated HuIFN-γ(78-92) but, at the same concentration, was not capable of inhibiting the nuclear import of APC-conjugated HuIFN-γ(122-132), suggesting that HuIFN-γ(78-92) was a less efficient NLS than HuIFN-γ(122-132). This is consistent with >90% loss of antiviral activity of HuIFN-γ lacking the downstream NLS in 122-132. The nuclear import of APC-conjugated HuIFN-γ(122-132) was inhibited by a peptide containing the prototypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/importin cellular machinery is used in both cases.
There appears to be strong conservation of the NLS motif as a mechanism for nuclear localization. Evolution seemed to have used part of the existing DNA-binding mechanism when compartmentalizing DNA-binding proteins into the nucleus. Cokol, M., et al., EMBO Rep. (2000), 1(5), 411-415 estimate that greater than 17% of all eukaryotic proteins may be imported into the nucleus, and after analyzing a set of 91 experimentally verified NLSs from the literature and expanding this set to 214 potential NLSs through iterated “in silico mutagenesis”. This final set matched in 43% of all known nuclear proteins and in no known non-nuclear protein. Cokel et al found an overlap between the NLS and DNA-binding region for 90% of the proteins for which both the NLS and DNA-binding regions were known, but only 56 of the 214 NLS motifs overlapped with DNA-binding regions. These 56 NLSs enabled a de novo prediction of partial DNA-binding regions for approximately 800 proteins in human, fly, worm and yeast.
More recently, it has been reported that NLS signal peptide can induce structural changes of DNA. The plant enzyme, glutaminyl-tRNA synthetase (GlnRS) from Lupinus luteus, contains an NLS at the N-terminal, a lysine rich polypeptide, KPKKKKEK (SEQ ID NO:8) Krzyzaniak, A., et al. Mol. Biol. Rep. (2000), 27(1), 51-54. Two synthetic peptides (20 and 8 amino acids long), derived from the NLS sequence of lupin GlnRS interact with DNA. In addition, the shorter 8 amino acid peptide caused the DNA to change its conformation from the B to the Z form. This observation clearly suggests that the presence of the NLS polypeptide in a leader sequence of GlnRS is required not only for protein transport into nucleus but also for regulation of a gene expression. This is the first report suggesting a role of the NLS signal peptide in structural changes of DNA.
Typically there is strong conservation of the NLS sequence within species. For example, the NLS in the N-terminal region of Smad 3 protein, the major Smad protein involved in TGF-β signal transduction, has a basic motif Lys40-Lys-Leu-Lys-Lys44 (SEQ ID NO:9), which is conserved among all the pathway-specific Smad proteins, and is required for Smad 3 nuclear import in response to ligand. Smad proteins are intracellular mediators of transforming growth factor-β (TGF-β) and related cytokines. Xiao, Z., et al., J. Biol. Chem. (2000), 275(31), 23425-23428 identified the role the NLS plays in nuclear localization. The authors demonstrated that the isolated Smad 3 MH1 domain displays significant specific binding to import in β, which is diminished or eliminated by mutations in the NLS. Full-size Smad 3 exhibits weak but specific binding to importin β, which is enhanced after phosphorylation by the type 1 TGF-β receptor. In contrast, no interaction was observed between importin α and Smad 3 or its MH1 domain, indicating that nuclear translocation of Smad proteins may occur through direct binding to importin β. The authors conclude that activation of all of the pathway-specific Smad proteins (Smads 1, 2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then binds directly to importin β triggers nuclear translocation.
In all cells, the lipid bilayer of cell membranes serves as a selective barrier for the passage of charged molecules, with the internalization of hydrophilic macromolecules being achieved through classical transport pathways (Hawiger, J., Curr Opin Chem Biol. 3, 89-94 (1999), Schwarze, S. R., et al., Trends in Cell Biology 10, 290-295 (2000)). These classical mechanisms of internalization involve receptor-mediated endocytosis or transporter dependent uptake (Cleves, A. E., Current Biology 7, R318-R320 (1997)). In contrast, an increasing number of molecules have been discovered that lack classical import and/or export signals (Cleves, A. E., Current Biology 7, R318-R320 (1997)). These molecules gain direct access to either cytoplasmic or nuclear compartments using unconventional processes of which the mechanisms remain largely unknown. These novel mechanisms are generally termed “nonclassical” and refer to transport pathways being used that are atypical. Relevant examples of this latter type are found in the gene-encoded proteins of HIV-1 TAT (Frankel, A. D. and Pabo, C. O. Cell 55, 1189-1193 (1988)), herpes virus VP22 (Elliott, C. and O'Hare, P. Cell 88, 223-233 (1997)), and Antennapedia, Antp (Derossi, D., et al., J. Biol. Chem. 269, 10444-10450 (1994)). It is now well established that the full-length proteins of HIV-1 TAT (Helland D. E., et al., J Virol 65, 4547-4549 (1991)), and VP22 (Pomeranz L. E. and Blaho J. A., J Virol 73, 6769-6731 (1999)) rapidly translocate into and out of cellular membranes. In fact, distinct peptide regions have been identified within both of these proteins that are capable of translocating into cellular compartments either alone or in combination with chimeric cargo peptides, and proteins (Lindgren, M, et al., Trends Pharmacol Sci. 3, 99-103 (2000), Derossi, D., et al, Trends Cell Biol., 8, 84-87 (1998), Prochiantz A., Current Opinion in Cell Biology 12, 400-406 (2000), Steven R. Schwarze, S. R., et al., Trends in Cell Biology 10, 290-295 (2000)). In contrast, full-length Antp protein has not been shown to traverse biological membranes; however, a 16 amino acid synthetic peptide derived from within its coding region does possess potent membrane penetrating abilities (Derossi, D., et al, Trends Cell Biol., 8, 84-87 (1998)). The accepted view of atypical transport used by these molecules has been termed “transduction” (Schwarze, S. R., et al. Trends in Cell Biology 10, 290-295 (2000)), and is currently defined as an extremely rapid membrane transport pathway that is receptor and energy independent, and can occur at 4° C. in all cell types (Schwarze, S. R. and Dowdy, S. F. Trends Pharmacol. Sci. 21, 45-48 (2000)). Interestingly, these three proteins are all nuclear proteins involved in transcriptional regulation, and their respective transducing peptides consist of strings of amino acids rich in arginine and lysine (Lindgren, M., et al., Trends Pharmacol Sci. 3, 99-103 (2000). Schwarze, S. R. and Dowdy, S. F. Trends Pharmacol. Sci. 21, 45-48 (2000)). However, irrespective of these similarities, these transducing peptides possess many different characteristics such as amino acid sequence, length of the sequence, cellular localization, and potency of membrane penetration. Thus, though each transducing sequence can penetrate cells and tissues, it has not been established whether they use the identical atypical transport mechanisms.
Finally, U.S. Pat. No. 6,022,950 teaches the use of a hybrid molecule of a portion of the binding domain of a cell-binding polypeptide ligand effective to cause said hybrid protein to bind to a cell of an animal, a translocation domain of naturally occurring protein which translocates said third part across the cytoplasmic membrane into the cytosol of the cell; and a chemical entity to be introduced into the cell. However, the patent teaches translocation domains of toxins. Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin and Pseudomonas exotoxin A, and may include other toxins and non-toxin molecules, as well. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al., Proc. Natl. Acad. Sci. USA 82:1692-1696, 1985; Colombatti et al., J. Biol. Chem. 261:3030-3035, 1986; and Deleers et al., FEBS 160:82-86, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al., Cell 48:129-136, 1987; and Gray et al., Proc. Natl. Acad. Sci. USA 81:2645-2649, 1984.
Given the considerable body of literature teaching control mechanisms of cellular localization, the proteins involved in regulation of intracellular transport, the different properties and control mechanisms for plasma membrane and the nuclear envelope, it is unexpected that polypeptides derived from mammalian proteins could transduce through the plasma membrane using nonclassical mechanisms and thus could be useful as membrane penetrating peptides useful as in vitro, ex vivo and in vivo delivery devices of a compound of interest. There is also considerable literature teaching non-protein derived methods for delivering a compound of interest into cells, for example electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, infection with modified viral nucleic acids, and direct microinjection into single cells, usually ova and the like. Each of these methods is relatively inefficient, resulting in relatively low percentage of the cells containing the delivered compound of interest and most of the methods are clearly not capable of realistic in vivo delivery. Many of the methods are toxic to the cells, resulting in relatively high apoptosis. Therefore, there is a considerable need for simple and more efficient delivery of compounds of interest into cells.